Summary Malignant melanoma in advanced stages is a highly lethal form of cancer, refractory to chemo- and radio-therapy, and the median survival is typically less than 4years. To improve response rates, antibodies that target CTL-4, PD1, and PDL1 has gained prominence, and can achieve a response rate of ~50% in metastatic cases. This is promising but a large proportion of patients still do not respond to immune therapies due to the absence of infiltrating T-cells, and presence of aberrant and suppresive signaling pathways that blunt the expression of checkpoint proteins (e.g. CD47, PDL1). To promote inflamed melanoma microenvironment, recent studies indicate that image-guided focused ultrasound (FUS) can i) mediate precise mechanical perturbation and elevation of tumor temperature to induce tumor antigen release and ii) upregulate calreticulin (CRT), a protein that is key to the activation of local and systemic anti-immunity. However, the exact mechanisms and how to translate this approach for clinical treatment of malignant melanoma is poorly understood. The goal of this project is to combine ultrasound guided FUS with novel CRT-NP, a liposome-plasmid nanoparticle agent that transfects melanoma cells to induce expression of CRT. Our in vitro and in vivo data in murine melanoma suggest that combined local treatment with CRT-NP/FUS (CFUS) enhances expression of CRT and modulates innate (CD47) and adaptive checkpoint proteins (PDL1); all of which significantly enhance the local and systemic immune priming and anti-tumor responses. Based on this premise, our central hypothesis is that CFUS targeted optimization of the CRT/CD47/PDL1 axis will provide powerful immune priming and generation of systemic immunity against malignant melanoma. To test our hypothesis, we will mechanistically dissect and understand CFUS-mediated immune priming in a B16 orthotopic and genetically-engineered mouse melanoma model (Aim 1 & 2) and translate this approach to clinical use in trials using client-owned dogs with spontaneous oral melanoma (Aims 3). Specifically, we will evaluate the impact of the CFUS treatment sequence, FUS exposures, and CRT-activation mechanisms in murine melanoma and translate this information to improve efficacy of checkpoint blockage in murine and canine tumor models. We expect that the successful optimization of local CFUS in this project will liberate tumors from their immune- suppressive state, achieve consistent and predictable clonal expansion of cytotoxic immune cells, and improve immunotherapy efficacy independent of cancer complexity. If successful, this method will provide a promising new avenue for treating melanoma and other types of solid tumor (e.g., breast, prostate) by significantly overcoming current immunotherapy barriers.